Our long-term goal is to understand transcription mechanism and regulation in molecular detail and to uncover underlying structural determinants. We focus on bacterial RNA polymerase (RNAP), the enzyme of transcription and a validated antibiotic target. During the previous funding period, crystallizable recombinant RNAP from Thermus aquaticus was obtained. This advance allows, for the first time, to combine the powers of genetic and structural approaches to understand RNAP function and regulation. During the tenure of this proposal, we will use available structural information and crystallizable wild-type and mutant recombinant RNAPs from Thermus and other thermophilic bacteria that we plan to obtain to address the following specific aims. 1. Study non-canonical RNAP-promoter DNA interactions during transcription initiation. The Thermus RNAP ? subunit residues involved in recognition of a novel element downstream of the -10 consensus promoter element will be identified and the functional and structural role of this element in promoter complex formation will be established, the role of the RNAP ?' subunit zipper structural element in promoter complex formation through interactions with promoter spacer will be determined, and the rules that govern the specific recognition of single-stranded M13 phage ori DNA by RNAP and the mechanism of primer RNA synthesis will be established. 2. Study RNAP interactions with antibiotics. Using site-specific suppression and Microcin J25 (MccJ25) mutants, the interaction of MccJ25 molecule with its binding site, the RNAP secondary channel, will be modeled. Structural homologues of MccJ25 that inhibit Gram-positive bacteria and target RNAPs from these organisms will be characterized. Functional and structural analyses of thermophilic RNAPs carrying site-specific mutants will be used to i) understand the differential effects of binding site mutations on RNAP interactions with Sorangicin and Rifampicin and ii) to understand the molecular mechanism of transcription inhibition by Streptolydigin. Mutational analysis of the RNAP proofreading center will be undertaken. The results of proposed studies will lead to new important insights into eubacterial RNAP mechanism and regulation and also into the mechanism of eukarytic RNAP function (this enzyme is highly similar to the eubacterial one). The proposed work is significant from the public health standpoint since information necessary for development of new and/or improved drugs that target bacterial transcription will be obtained. [unreadable] [unreadable] [unreadable]